Construction method and application of nucleic acid transporter based on yeast beta glucan nanoparticles

Abstract

The invention belongs to the technical field of biological materials, and provides a construction method and application of a nucleic acid transporter based on yeast beta glucan nanoparticles, beta glucan from yeast is cross-linked with 1.8 k PEI through carboxymethylation modification to prepare CMG-PEI NP to load nucleic acid, the nucleic acid transporter is applied to RNA transport, and a new way and means are provided for RNA transport. According to the invention, the structures of the CMG-PEINP and related components are evaluated, manninotriose is used for further wrapping the CMG-PEINP, a cell model is established, and the RNA transport efficiency of the CMG-PEINP transporter is evaluated. Results show that CMG-PEINP and nucleic acid can form a compound with a compact structure, the particle size meets the drug delivery requirement, and when the mass ratio of CMG-PEINP to eGFP mRNA is 48: 1, the transfection efficiency is the highest.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, and relates to yeast β-glucan nanoparticles, specifically to a method for constructing and applying gene transport vectors based on yeast β-glucan nanoparticles. Background Technology

[0002] The chemical structure of RNA alone is susceptible to degradation by nucleases. During circulation in the bloodstream, naked RNA rapidly decomposes, hindering its effective delivery to target tissues. Furthermore, RNA carries a negative charge, making it difficult to penetrate cell membranes. Therefore, carriers are needed for protection and assistance. With the rapid development of nanotechnology, nanocarriers have gradually become an important research direction in drug delivery. Nanoparticles possess many advantages, including small size (typically <200 nm), large specific surface area, and strong surface modifiability. They can encapsulate drugs or nucleic acids with high efficiency, improved stability, and strong support for targeted delivery. Currently common nanocarriers include liposomes, inorganic nanoparticles, and polymer nanoparticles. Liposomes were among the earliest nanocarriers used clinically. Similar to cell membranes, they have a bilayer phospholipid structure, enabling them to effectively encapsulate hydrophilic or hydrophobic drugs, thus achieving a slowed release effect. Inorganic nanoparticles (such as mesoporous silica and gold nanoparticles) have shown excellent efficacy in combination therapies. Mesoporous silica nanoparticles, in particular, possess a huge specific surface area and a regular pore structure, enabling them to efficiently load large amounts of drugs. Polymer nanoparticles (such as polyethyleneimine, PEI, used in this invention) have become popular materials for nanoparticle carriers in recent years due to their good biocompatibility and controllable degradation. PEI has an extremely high amino density, which enables it to effectively compress and protect nucleic acids. However, the selectivity of PEI in terms of cytotoxicity and transport efficiency limits its clinical application. To reduce PEI toxicity and improve delivery efficiency, researchers often chemically modify PEI, for example, by combining it with polysaccharides. With their excellent characteristics such as high drug loading capacity and photothermal effects, composite nanoparticles based on natural polysaccharides (such as dextran and chitosan) have become a research hotspot in recent years, exhibiting extremely high biocompatibility. Generally, the transfection efficiency of PEI is positively correlated with its cytotoxicity. Although low molecular weight PEI has low toxicity, its transport efficiency is low. In order to resolve the contradiction between the transport efficiency and cytotoxicity of PEI gene vectors, grafting modification is used to graft low molecular weight PEI onto the main chain of a high molecular weight polymer. This allows the grafted polymer to both compress nucleic acids well and have a strong proton buffering capacity, thereby improving the gene transport capacity of low molecular weight PEI.

[0003] Dextran is a natural polysaccharide often used as an excellent material for constructing drug delivery systems. Yeast dextran is a polysaccharide with β-1,3-glucose as the main chain and β-1,6-glucose as the branch chain. Its molecule contains a large number of hydroxyl groups (-OH) and possesses excellent degradability, biocompatibility, and low immunogenicity. Studies have shown that carboxymethylated β-glucan (CMG) can be chemically cross-linked with PEI to form composite nanocarrier particles (CMG-PEI NP). These nanoparticles utilize the good biocompatibility and protective compression ability of CMG for nucleic acids, synergistically with the "proton sponge effect" of low molecular weight PEI, to solve the problem of the CMG-nucleic acid complex's difficulty in escaping into the endosome, thus obtaining a highly efficient and low-toxicity gene transport vector. Therefore, CMG-PEI NP material is an advantageous carrier for RNA delivery. In addition, the abundant amino groups on the surface of CMG-PEI NP can be functionalized (such as reacting with the aldehyde groups of mannotriose to undergo an amine-aldehyde reaction), gradually improving the stability of the particles and enhancing their targeting or immunomodulatory functions. For example, aldehyde-coated CMG-PEI NPs can significantly improve antigen presentation efficiency because they can target dendritic cells (DCs) via the mannose receptor. Compared to chitosan-PEI complexes, CMG-PEI NPs are easier to prepare on a large scale because they contain a neutral charge. Compared to lipid nanoparticles, CMG-PEI NPs do not require complex cold chain storage, making them suitable for environments with relatively limited resources and more appropriate for research aimed at clinical applications. Furthermore, the dextran groups offer more possibilities for research into multifunctional modifications.

[0004] Based on the above research, this invention provides a method for constructing a yeast-derived carboxymethylated dextran transporter, preparing CMG-PEI NP for nucleic acid adsorption, and performing cell transfection experiments on the nucleic acid-loaded CMG-PEI NP to verify its transport capacity, thus providing a new pathway and means for nucleic acid transport. Summary of the Invention

[0005] The purpose of this invention is to provide a method and application for constructing a gene transport vector based on yeast β-glucan nanoparticles. A CMG-PEI NP complex is prepared by crosslinking carboxymethyl glucan with 1.8k PEI, which can load nucleic acids. This complex is then applied to RNA transport, providing a new pathway and method for RNA transport. This addresses the problems of RNA's chemical structure being easily degraded by nucleases, the potential for immune responses to naked RNA circulating in the blood, and the negative charge of RNA repelling the cell membrane, thus preventing direct entry and requiring a carrier for protection and assistance in penetrating the cell membrane.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] This invention provides a method for constructing a nucleic acid transport vector based on yeast β-glucan nanoparticles, comprising the following steps:

[0008] a. Fractional alcohol precipitation of a 1% carboxymethyl dextran solution using anhydrous ethanol;

[0009] b. The carboxymethyl dextran after precipitation and separation was cross-linked with 1.8k PEI through chemical bonds using the EDC / NHS cross-linking method to form CMG-PEI NP;

[0010] c. Combine CMG-PEI NP with nucleic acid to form CMG-PEI-nucleic acid complex;

[0011] d. The CMG-PEI-nucleic acid complex was encapsulated with mannotriose to form a mannotriose-encapsulated CMG-PEI-nucleic acid transport vector.

[0012] Preferably, the precipitation separation specifically involves: adding anhydrous ethanol dropwise to the carboxymethyl dextran solution until a partial white precipitate appears, at which point the volume percentage of anhydrous ethanol added is 40%. Separation is then performed to obtain a first supernatant and a first precipitate. Anhydrous ethanol is then added dropwise to the first supernatant until the volume fraction of ethanol is 50%. Separation is then performed to obtain a second supernatant and a second precipitate. Anhydrous ethanol is then added dropwise to the second supernatant until the volume fraction of ethanol is 60%. Separation is then performed to obtain a third supernatant and a third precipitate. Thus, the carboxymethyl dextran solution is separated into four molecular weight carboxymethyl dextrans corresponding to the first precipitate, second precipitate, third precipitate, and third supernatant.

[0013] Preferably, the crosslinking method is as follows: EDC and NHS are dissolved in 20 mM MES activation buffer, then mixed with CMG and reacted under magnetic stirring for 30 min. Then, 1.8 kJ of fully dissolved PEI is added dropwise. Tween 80 is added to the above mixed solution. The pH is adjusted to 5-6 using 1 M HCl. An appropriate amount of anhydrous ethanol is slowly added dropwise until the solution becomes turbid. The reaction is stopped after 50 min. The crosslinking reaction is terminated with 1 M TRIS-HCl at pH 8. Dialysis is performed in a dialysis bag at 8-14 K for 48 h.

[0014] Preferably, the volume ratio of Tween 80 to water solvent is 12:1100, and the mass ratio of EDC, NHS, CMG, and PEI is 5:5:8:7.

[0015] Preferably, the binding of CMG-PEI NP to nucleic acid specifically includes: adding an aqueous solution of CMG-PEI NP to an equal mass of RNA solution, vortexing for 30 seconds, and incubating at room temperature for 30 minutes to obtain a CMG-PEI-nucleic acid complex solution, wherein the mass ratio of CMG-PEI NP to RNA is in the range of 1:1 to 10:1.

[0016] Preferably, the mass ratio of CMG-PEI NP to RNA is 6:1 to 7:1.

[0017] The present invention also provides a nucleic acid transport vector based on yeast β-glucan nanoparticles, which is prepared by the above-described gene transport vector construction method.

[0018] This invention also provides the application of the above-mentioned nucleic acid transport vector in transporting nucleic acids. The CMG-PEI NP obtained above is complexed with RNA, and the CMG-PEI-nucleic acid complex is encapsulated with mannotriose to form a nucleic acid transport vector for transporting RNA.

[0019] Preferably, the preparation of the CMG-PEI-nucleic acid complex gene transport vector specifically includes: mixing eGFP mRNA with CMG-PEI NP, vortexing vigorously for about 30 seconds, incubating at room temperature for about 30 minutes, adding the CMG-PEI NP and nucleic acid complex to mannotriose, with a final concentration of 4 mg / mL, mixing well, and reacting overnight for 12 hours, with a CMG-PEI NP:eGFP mRNA mass ratio of 16:1 to 48:1.

[0020] Preferably, the mass ratio of CMG-PEI NP:eGFP mRNA is 48:1.

[0021] The beneficial effects of this invention are:

[0022] This invention successfully prepared CMG-PEI NPs by crosslinking carboxymethyl dextran with 1.8k PEI. The combination with RNA demonstrates that CMG-PEI NPs can load nucleic acids, providing a novel pathway and method for RNA transport. The structure of CMG-PEI NPs and related components was evaluated. CMG-PEI NPs were further encapsulated using mannotriose, and a cell model was established to assess the efficiency of CMG-PEI-nucleic acid transport vectors in transporting RNA. The results show that CMG-PEI nanoparticles can form relatively compact complexes with nucleic acids, and the particle size meets the requirements for drug delivery. Attached Figure Description

[0023] Figure 1The diagram shows the molecular weight and conformation of the four polysaccharides in this invention (A is CMG-PEINP corresponding to the first precipitate; B is CMG-PEINP corresponding to the second precipitate; C is CMG-PEINP corresponding to the third precipitate; D is CMG-PEINP corresponding to the third supernatant).

[0024] Figure 2 These are the transmission electron microscopy observation results of CMG-PEI in this invention (A is the CMG-PEI NP corresponding to the first precipitate; B is the CMG-PEI NP corresponding to the second precipitate; C is the CMG-PEI NP corresponding to the third precipitate; D is the CMG-PEI NP corresponding to the third supernatant).

[0025] Figure 3 The results of agarose gel electrophoresis of CMG-PEI NP-loaded RNA in this invention are as follows (in A, L0: nucleic acid marker, L1-L5: mass ratio 0.2:1, 0.4:1, 1:1, 10:1, 20:1; in B, L0: RNA control, L1-L7: mass ratio 2:1 to 8:1).

[0026] Figure 4 These are the electron microscopy observation results of the CMG-PEI-nucleic acid complex corresponding to the third precipitate encapsulated by mannotriose in this invention (A is a 200× magnified image; B is a 400× magnified image).

[0027] Figure 5 These are the CMG-PEI NP infrared characterization results in this invention;

[0028] Figure 6 This invention includes dynamic light scattering analysis (A shows the zeta potential analysis results of CMG-PEI NP and CMG-PEI-nucleic acid complex; B shows the particle size analysis results of CMG-PEI NP and CMG-PEI-nucleic acid complex).

[0029] Figure 7 This is the cell viability diagram in this invention (A represents 12 h; B represents 24 h; different letters indicate significant differences).

[0030] Figure 8 The fluorescence observation diagrams in this invention are as follows: (A: 16:1 without mannotriose; B: 16:1 with mannotriose; C: 24:1 without mannotriose; D: 24:1 with mannotriose; E: 32:1 without mannotriose; F: 32:1 with mannotriose; G: 40:1 without mannotriose; H: 40:1 with mannotriose; I: 48:1 without mannotriose; J: 48:1 with mannotriose; K: 16:1 corresponding to 1.8k PEI control; L: 48:1 corresponding to 1.8k PEI control; 200×). Detailed Implementation

[0031] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0032] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0033] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0034] Example

[0035] 1. Construction of transport carrier

[0036] 1.1 Alcohol precipitation separation of carboxymethyl dextran of different molecular weights

[0037] 10 g of commercially available carboxymethyl dextran was weighed and dissolved in 200 mL of deionized water. The solution was filtered through multiple layers of filter screens, including a 0.7 μm filter membrane and a 0.45 μm filter membrane to remove the insoluble portion of the carboxymethyl dextran. Anhydrous ethanol was added dropwise to the carboxymethyl dextran solution until a partial white precipitate appeared. At this point, the volume percentage of anhydrous ethanol added was 40%. The solution was separated to obtain the first supernatant and the first precipitate. Anhydrous ethanol was added dropwise to the first supernatant until the volume fraction of ethanol was 50%. The solution was separated to obtain the second supernatant and the second precipitate. Anhydrous ethanol was added dropwise to the second supernatant until the volume fraction of ethanol was 60%. The solution was separated to obtain the third supernatant and the third precipitate. Thus, the carboxymethyl dextran solution was separated into four molecular weights of carboxymethyl dextran corresponding to the first precipitate, the second precipitate, the third precipitate, and the third supernatant.

[0038] The molecular weight and conformational distribution of carboxymethyl dextran in the first, second, and third precipitates and the third supernatant can be accurately determined using high performance liquid chromatography-size exclusion chromatography coupled with laser light scattering and differential detector (HPSEC-MALLS-RI). The experimental steps are as follows:

[0039] (1) Sample solution preparation

[0040] Accurately weigh 10.0 mg of each polysaccharide sample, and dilute to 1.0 mL to prepare a 10.0 mg / mL solution. -1 The sample solutions were filtered through a 0.22 μm microporous membrane and then analyzed by high performance liquid chromatography (HPLC) with size exclusion chromatography.

[0041] (2) Chromatographic conditions

[0042] Chromatographic columns: OHpak SB-806 HQ 300×8 mm, OHpak SB-804HQ 300×8 mm (7.9 mm×300 mm, Shodex, Japan) in tandem; mobile phase: 0.2 M NaCl; flow rate: 1.0 mL·min⁻¹; detectors: (RID-G7162A) differential detector and DAWN detector (Wyatt Technology Co., USA);

[0043] The weight-average molecular weight and number-average molecular weight (Mw and Mn) and polydispersity index (Mw / Mn) were determined. ASTRA 8 software was used for data collection and calculation.

[0044] 1.2 CMG-PEI NP Crosslinking Steps

[0045] Weigh 40 mg of carboxymethyl dextran (the cross-linking reaction is carried out according to mass; the experimental conditions are the same for the four different molecular weights of carboxymethyl dextran except for the molecular weight of the sugar). Dissolve in 5 mL of deionized water and place in a 50 mL beaker. Under magnetic stirring, prepare 20 mM MES activation buffer (0.018023 corresponds to 5 mL, which can be prepared in centrifuge tubes). At the same time, weigh 0.025 g each of EDC and NHS and place them in centrifuge tubes. Dissolve EDC and NHS in 2.5 mL of the prepared MES activation buffer and mix with CMG. React under magnetic stirring for 30 min to activate the carboxymethyl groups of carboxymethyl dextran. During this process, weigh a small amount of 1.8 kPa PEI (35 mg) using an electronic balance and dissolve it in 1 mL of water. After the activation process is complete, first add the fully dissolved 1.8 kPa PEI, then add 120 μL of Tween 80 to avoid flocculation during the reaction. Quickly use 1 M... Adjust the pH to 5-6 with HCl, slowly add an appropriate amount of anhydrous ethanol, and stop when the mixture becomes slightly turbid. The amount of ethanol added varies depending on the concentration of carboxymethyl dextran used for precipitation. React under magnetic stirring for 50 min, and terminate the cross-linking reaction with 1M TRIS-HCl at pH 8. Select a dialysis bag at 8-14K ​​and boil it with deionized water for 30 min to activate the dialysis bag. Then transfer the sample to the dialysis bag and dialyze for 48 h to remove excess unreacted EDC / NHS and 1.8k PEI.

[0046] 1.3 Transmission electron microscopy observation results of CMG-PEI NPs with different molecular weights

[0047] Four different molecular weight CMG-PEI NPs crosslinked with carboxymethyl dextran (crosslinked according to the method in 1.2 of this example) were filtered through a 0.45 μm microporous membrane, and an appropriate amount was dropped onto a carbon support membrane and allowed to dry naturally at room temperature. The morphology of the CMG-PEI NP complex was observed using transmission electron microscopy (TEM). The observation of the CMG-PEI NP samples was performed on a JEM-2100 transmission electron microscope manufactured by Nippon Electronics, with an accelerating voltage of 80–200 kV.

[0048] For TEM sample preparation, a small amount of thoroughly mixed CMG-PEI NP solution is dropped onto an ultrathin carbon support film copper mesh and allowed to dry. The copper mesh with the sample on it is then placed in a plasma cleaner and plasma-cleaned at 15 W for 50 s. After plasma cleaning, the copper mesh should be immediately examined under an electron microscope.

[0049] 1.4 Gel electrophoresis detection of CMG-PEI NP-loaded nucleic acids

[0050] A 1 mg / mL CMG-PEI NP solution was prepared using deionized water as the solvent and filtered through a 0.45 μm microporous membrane. Different volumes of CMG-PEI NP aqueous solution were added to equal volumes of RNA solution, vortexed for 30 s, and incubated at room temperature for 30 min to obtain CMG-PEI-nucleic acid complex solutions with different mass ratios. By adding different volumes of CMG-PEI NP solution to 1 μg of RNA, a large gradient preparation was first performed to confirm the encapsulation range, followed by a small gradient preparation to refine the encapsulation. Specifically, CMG-PEI-nucleic acid complexes with mass ratios of 0.2:1, 0.4:1, 1:1, 10:1, and 20:1 were prepared. A 1% agarose gel was prepared using TAE electrophoresis buffer, with ethidium bromide added as a chromogenic agent. Each complex was added to a well of the gel, with blank RNA as a control. The electrophoresis voltage was set to 70 V, the electrophoresis time to 30 min, and images were captured using a gel imaging system. After confirming the ratio range based on the photographic records, the gradient difference was reduced and the agarose gel electrophoresis was repeated.

[0051] 1.5 Transmission electron microscopy observation of CMG-PEI-nucleic acid complex encapsulated by mannotriose

[0052] Considering the particle size of CMG-PEI NP as a transport carrier, a suitable CMG-PEI NP was selected for the amine-aldehyde reaction with mannotriose. The specific steps were as follows: 0.5 μg of eGFP mRNA (concentration of 1 mg / mL, eGFP mRNA is the messenger RNA encoding enhanced green fluorescent protein) and the corresponding CMG-PEI NP (CMG-PEI NP concentration of 2 mg / mL) in the third precipitate were taken, vortexed vigorously for about 30 s, and incubated at room temperature for about 30 min. The CMG-PEI-nucleic acid complex was then added to 500 μg of mannotriose (with aldehyde group) (mannotriose stock solution concentration of 20 mg / mL), mixed well, and reacted overnight for 12 h. The unreacted part was removed by dialysis, and the mannotriose-encapsulated part was observed again by transmission electron microscopy.

[0053] Finally, based on the electron microscopy results, the CMG corresponding to the third precipitate with uniform particle size and appropriate size was cross-linked with PEI and then subjected to infrared characterization, particle size measurement, zeta potential measurement, and subsequent cell experiments.

[0054] 1.4 Infrared Detection

[0055] Whether CMG-PEI NP has been successfully cross-linked is determined by conventional infrared detection.

[0056] 1.5 Dynamic Light Scattering Analysis

[0057] Dynamic light scattering analysis was used to analyze the particle size and zeta potential of CMG-PEI NP and CMG-PEI-nucleic acid complex to determine the stability of CMG-PEI NP binding to nucleic acid molecules.

[0058] 2. Experimental Results

[0059] 2.1 Molecular weight and conformation of polysaccharides

[0060] according to Figure 1 The results showed that the retention times of the four carboxymethyl dextrans separated by alcohol precipitation were all between 15 and 25 min, indicating that they were the same substance. The precipitate after precipitation with 40% ethanol (v / v) changed from a bimodal to a single, more uniform peak in the supernatant after precipitation with 60% ethanol (v / v), indicating that alcohol precipitation can improve the purity of carboxymethyl dextran. The number-average molecular weight (Mn) of the third precipitate was 1.801 × 10⁻⁶. 4 The weight-average molecular weight (Mw) is 2.252 × 10⁻⁶. 4 Its polydispersity index (Mw / Mn) is 1.250, and its PDI is close to 1, indicating that its molecular weight distribution is uniform and its surface composition is simple. Furthermore, the molecular weight shows a single peak around 20 min, which also proves that the sample is relatively uniform and has a simple composition.

[0061] 2.2 Transmission electron microscopy observation results of CMG-PEI NPs with different molecular weights

[0062] Transmission electron microscopy results show that ( Figure 2 The particle size of four different molecular weight carboxymethyl dextrans crosslinked with 1.8k PEI was consistent with the relative molecular weight of the carboxymethyl dextran, indicating a strong positive correlation between the hydrodynamic diameter and molecular weight of the carboxymethyl dextran. For the CMG-PEI NPs crosslinked from the precipitate after precipitation with 40% ethanol to the supernatant after precipitation with 60% ethanol, the particle size decreased sequentially, and the size of the CMG-PEI NPs was positively correlated with the original molecular weight of CMG. The third precipitate showed a suitable and uniform relative particle size for the CMG-PEI NPs.

[0063] 2.3 Gel electrophoresis results

[0064] The CMG-PEI NP corresponding to the third precipitate was complexed with RNA, and its binding ability to nucleic acids was determined by agarose gel electrophoresis. Negatively charged nucleic acids can migrate from the negative electrode to the positive electrode through the agarose gel under the influence of an electric field. When the cationic polymer is mixed with nucleic acid, the cationic polymer encapsulates and compresses the nucleic acid, neutralizing some or all of the negative charge and inhibiting electrophoresis. Ethidium bromide (EB) can insert into the nucleic acid chain and exhibits strong fluorescence under ultraviolet light, while almost no fluorescence is observed in its free state. Therefore, by observing the position and intensity of the fluorescent bands in the gel electrophoresis experiment, the binding status of the cationic polymer to nucleic acid can be determined, thereby qualitatively confirming the binding ability of CMG-PEI NP to nucleic acids.

[0065] The results showed that ( Figure 3 As shown in A, at low mass ratios (0.2:1, 0.4:1), the RNA band was brighter, indicating that CMG-PEI NP failed to effectively bind RNA. At a mass ratio of 1:1, the RNA band brightness decreased slightly, and a slight retention phenomenon began to appear, indicating that CMG-PEI NP and RNA partially bound. At high mass ratios (10:1, 20:1), the RNA band disappeared significantly, and the complex remained near the sample well due to reduced migration rate caused by charge neutralization, indicating that CMG-PEI NP completely encapsulated the RNA. Therefore, the preliminary conclusion is that the effective payload mass ratio range is between 1:1 and 10:1, requiring further fine screening.

[0066] from Figure 3As shown in section B, when the mass ratio is 2:1-4:1, the brightness of the RNA band gradually decreases with increasing ratio. The CMG-PEI NP complex with nucleic acid begins to form but binding is incomplete, and some nucleic acid still escapes. When the mass ratio is 5:1-8:1, the RNA band significantly weakens, and obvious complex retention appears near the sample well. Furthermore, the 8:1 group shows almost no free RNA band. Therefore, the final conclusion is that when the mass ratio reaches 5:1-8:1, nanoparticles can efficiently bind RNA, with 6:1-7:1 being the optimal loading range (balancing binding efficiency and complex stability).

[0067] In summary, CMG-PEI NP possesses nucleic acid loading capacity.

[0068] 2.4 Transmission electron microscopy observation of CMG-PEI-nucleic acid complex encapsulated by mannotriose

[0069] Transmission electron microscopy results show that ( Figure 4 The third precipitate, corresponding to CMG-PEI NP, was slightly larger after encapsulating mannotriose, and the boundary was lighter in color than the interior. At the same time, a larger particle was selected and observed at a higher magnification. It was found that the boundary was surrounded by small particles, which is mannotriose encapsulation.

[0070] 2.5 Infrared Detection Results

[0071] Infrared detection results show that ( Figure 5 ): PEI is at 3300-3500 cm -1 There is an absorption peak, which is due to the NH stretching vibration. The PEI value is between 1600 and 1650 cm⁻¹. -1 There is an absorption peak, which is due to the NH bending vibration; PEI is at 1100 cm⁻¹. -1 There is an absorption peak nearby. This is due to the CN stretching vibration.

[0072] CMG at 3300-3500 cm -1 There are broad peaks, which are due to the stretching vibrations of OH and NH. Carboxymethyl dextran shows peaks at 1600-1700 cm⁻¹. -1 There is an absorption peak, which is due to the C=O stretching vibration. Carboxymethyl dextran has an absorption peak in the range of 1000-1200 cm⁻¹. -1 There are multiple peaks, which are due to the stretching vibrations of COC and COH.

[0073] CMG-PEI NP at 3300-3500 cm -1 The broad peaks are more pronounced within the range of 1600-1650 cm⁻¹, indicating the presence of amino (NH) and hydroxyl (OH) groups; -1 New or enhanced absorption peaks appear at 1300-1400 cm⁻¹, corresponding to the NH bending vibration of amide II and the C=O stretching vibration of amide I;-1 An absorption peak appears at [value missing], corresponding to the CN stretching vibration of amide III; [value missing] in the 1000-1200 cm⁻¹ range. -1 The presence of characteristic peaks of carboxymethyl dextran within the range indicates successful grafting of PEI.

[0074] By comparing the infrared spectra of the three materials, it can be concluded that carboxymethyl dextran was successfully synthesized: its spectrum shows characteristic peaks of carboxyl groups (C=O) and glycosidic bonds (COC), and PEI was successfully grafted onto carboxymethyl dextran. In the spectrum of CMG-PEI NP, vibrational peaks of NH and CN unique to PEI appeared. The newly appearing amide I and amide II peaks indicate that chemical bonding has occurred between PEI and carboxymethyl dextran.

[0075] 2.6 Results of Dynamic Light Scattering Analysis

[0076] Zeta potential analysis showed ( Figure 6 A) CMG-PEI NPs exhibit a high surface positive potential (+15~20mV) in aqueous solution, a characteristic that allows them to be loaded with nucleic acids and prevents particle sedimentation and aggregation, as well as addressing issues such as insufficient electrostatic repulsion. Furthermore, CMG-PEI NPs demonstrate high stability in practical applications.

[0077] According to the particle size analyzer (measured by the particle size analyzer) Figure 6 B), Particle size analysis showed that the CMG-PEI NP size was mostly distributed at 150 nm, while the particle size was reduced to 100 nm after nucleic acid compression, which meets the requirements for drug delivery.

[0078] Application Examples

[0079] 1. Cell experiments

[0080] 1.1 Basic Operations

[0081] Cell resuscitation

[0082] Remove frozen cells from liquid nitrogen tank → Thaw in 37°C water bath → Pipette 1 mL of culture medium into cryovial and mix well → Transfer cells to 15 mL centrifuge tube → Add 3 mL of culture medium to centrifuge tube → Centrifuge (1000 rpm / 5 min) → Prepare T25 cell culture flask and label it with: date, time, name, and cell name → Add 3-4 mL of culture medium to the labeled T25 culture flask → Remove the centrifuged cells and discard the supernatant in the centrifuge tube → Pipette 1 mL of culture medium into centrifuge tube and mix well → Transfer cells from centrifuge tube to the labeled culture flask → Mix well using the figure-eight or cross method → ​​Transfer to a 37°C, 5% CO2 incubator for culture.

[0083] Cell passage

[0084] Remove the culture flask from the incubator → Wash twice with 1 mL PBS → Remove the PBS → Add 1 mL of trypsin to the culture flask → Gently shake the flask to allow the trypsin to infiltrate the cells → Place the culture flask in the incubator for 1-2 min to accelerate digestion → Remove the culture flask and observe (most cells becoming round indicates sufficient digestion) → Add 2 mL of culture medium to stop digestion and mix well → Transfer the liquid from the culture flask to a 15 mL centrifuge tube → Centrifuge (800 rpm / 3 min) → Label the new culture flask (date, time, name, cell name) → Add culture medium to the new culture flask → Remove the centrifuge tube and discard the supernatant → Add 1 mL of culture medium and mix well → Transfer to the new culture flask → Mix well using the figure-eight and cross-hatching methods → Transfer to a 37°C, 5% CO2 incubator for culture.

[0085] Cell seeding plate

[0086] Remove the culture flask from the incubator → Wash twice with 1 mL PBS → Remove the PBS → Add 1 mL of trypsin to the culture flask → Gently shake the flask to allow the trypsin to infiltrate the cells → Place the culture flask in the incubator for 1-2 min to accelerate digestion → Remove the culture flask and observe (most cells becoming round indicates sufficient digestion) → Add 2 mL of culture medium to stop digestion and mix well → Transfer the liquid from the culture flask to a 15 mL centrifuge tube → Centrifuge (800 rpm / 3 min) → Label the new cell culture plate (date, time, name, cell name) → Add culture medium to the new culture flask → Remove the centrifuge tube and discard the supernatant → Calculate the cell number according to the required plate size and number of wells, add an appropriate amount of culture medium and mix well → Transfer to a new culture plate → Use different methods to mix wells for different plate sizes → Transfer to a 37°C, 5% CO2 incubator for culture.

[0087] 1.2 CCK8 assay for cytotoxicity

[0088] RAW264.7 cells were used for cell proliferation-toxicity assay in DMEM high-glucose complete medium. 96-well plates were required, and the culture plates were pre-cultured in an incubator for 24 hours (at 37°C and 5% CO2).

[0089] Add 10 μL of the test substance at different concentrations to the culture plate. This cytotoxicity assay requires five concentrations (0.08 mg / mL, 0.4 mg / mL, 2 mg / mL, 4 mg / mL, and 10 mg / mL) corresponding to 1.8 kDa PEI, CMG-PEI NP, and mannotriose-coated CMG-PEI NP, respectively. Three replicates are required for each well. Two sets of experiments are also required, incubated for 12 and 24 hours respectively.

[0090] Remove the culture medium and wash the cells twice with the medium, then add fresh medium to remove any drug interference. Add 10 μL of CCK-8 solution to each well (be careful not to generate air bubbles in the wells, as they will affect the OD reading). Continue to incubate the plate in an incubator for 1–4 h, specifically by measuring the OD at 450 nm every 0.5 h and recording the absorbance at 450 nm as measured by a microplate reader.

[0091] Vitality Calculation:

[0092] Cell viability (%) = [A(drug-treated) - A(blank)] / [A(0-drug-treated) - A(blank)] x 100%

[0093] A (Drug Addition): Absorbance of the pores containing cells, CCK-8 solution, and drug solution.

[0094] A (Blank): Absorbance of wells containing culture medium and CCK-8 solution but without cells.

[0095] A (0 drug added): Absorbance of the well containing cells and CCK-8 solution but no drug solution.

[0096] A bar chart is plotted based on the calculated cell viability.

[0097] 1.3 Cell transfection experiment

[0098] This experiment used two cell lines, 293a and RAW264.7. After the cells reached confluence, they were digested and seeded into 24-well plates. Based on the agarose gel electrophoresis results, different mass ratios of CMG-PEI NP and eGFP mRNA were prepared. Simultaneously, CMG-PEI-nucleic acid complexes encapsulated with mannotriose were prepared in corresponding ratios of 16:1, 24:1, 32:1, 40:1, and 48:1. The mass of eGFP mRNA was set to 2 μg (eGFP mRNA concentration of 1 mg / ml). For the 48:1 mass ratio, 96 μg of CMG-PEI NP was required, with a CMG-PEI NP concentration of 2 mg / ml, meaning a required CMG-PEI NP volume of 48 μL. Therefore, the CMG-PEI NP concentration at this point was 1.92 mg / ml. The same logic applied to other mass ratios. The mass of mannotriose added was calculated as ten times the mass of CMG-PEI NP (based on the number of bound amino and aldehyde groups).

[0099] Transfection experiments were initiated when cells reached 70% confluency in the wells. A blank control group, different mass ratios of CMG-PEI NPs, and their corresponding mannotriose-encapsulated CMG-PEI-nucleic acid transport vectors were set up. Note that when using the 1.8kJ PEI control loaded with nucleic acid, serum-free medium must be used; the medium should be added to the wells before mixing with complete medium. Transfection efficiency was observed using a fluorescence microscope 24 hours after transfection. The optimal mass ratio was determined, and the transfection efficiency was verified.

[0100] Using eGFP mRNA encoding enhanced green fluorescent protein, cells were transported and then placed directly onto the stage of an Olympus 1X51 inverted fluorescence microscope. The green fluorescent protein signal was observed and photographed (200×). Positive cells emitted bright green fluorescence, while negative cells showed no green fluorescence. The amount of cell fluorescence was observed to assess transport efficiency.

[0101] 1.4 Data Analysis

[0102] Data were analyzed using SPSS 23.0 software and expressed as mean ± SD. One-way ANOVA was used for comparisons among multiple groups. P < 0.05 was considered statistically significant. Alphabetical notation was used.

[0103] 2. Experimental Results

[0104] 2.1 Cell viability results

[0105] The results show that ( Figure 7 The drug, in the order of CMG-PEI NP, CMG-PEI NP, and 1.8k PEI encapsulated with mannotrisaccharide, caused the viability of RAW264.7 cells to decrease sequentially. This indicates that the cytotoxicity of the nanoparticles after encapsulating mannotrisaccharide is reduced, and it exhibits a significant concentration-dependent effect, meaning that the higher the drug concentration, the greater the cytotoxicity.

[0106] 2.2 Transfection Results

[0107] The results show that ( Figure 8 As the CMG-PEI NP:eGFP mRNA mass ratio increases, the transfection efficiency improves. Based on the cytotoxicity results, a 48:1 ratio is optimal. At this ratio, the same mass of 1.8k PEI in CMG-PEI NP can be observed to significantly improve cell transfection efficiency.

[0108] In summary, this invention successfully prepared CMG-PEI NP by crosslinking carboxymethyl dextran with PEI. The combination with RNA demonstrates that CMG-PEI NP can load nucleic acids, with the optimal mass ratio of CMG-PEI NP to RNA being 6:1-7:1. The structure of CMG-PEI NP and related components was evaluated. CMG-PEI NP was further encapsulated using mannotriose, and a cell model was established. CMG-PEI NP was then applied to RNA transport to assess the efficiency of CMG-PEI-nucleic acid transport vectors in transporting RNA. The results show that CMG-PEI NP can form a relatively tight complex with nucleic acids, with a particle size suitable for drug delivery. The highest transfection efficiency was achieved when the mass ratio of CMG-PEI NP to eGFP mRNA was 48:1.

[0109] The above-described embodiments are merely preferred embodiments of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the appended claims.

Claims

1. A method for constructing a nucleic acid transport vector based on yeast β-glucan nanoparticles, characterized in that, Includes the following steps: a. Fractional alcohol precipitation of a 1% carboxymethyl dextran solution using anhydrous ethanol; b. The carboxymethyl dextran after fractional alcohol precipitation and 1.8k PEI were cross-linked by chemical bonds using the EDC / NHS cross-linking method to form CMG-PEI NP, i.e., CMG-PEI NP; c. Combine CMG-PEI NP with nucleic acid to form CMG-PEI-nucleic acid complex; d. The CMG-PEI-nucleic acid complex was encapsulated with mannotriose to form a mannotriose-encapsulated CMG-PEI-nucleic acid transport vector.

2. The construction method according to claim 1, characterized in that, The fractional alcohol precipitation is specifically as follows: anhydrous ethanol is added dropwise to the carboxymethyl dextran solution until a partial white precipitate appears. At this point, the volume percentage of anhydrous ethanol added is 40%. The solution is then separated to obtain a first supernatant and a first precipitate. Anhydrous ethanol is continued to be added dropwise to the first supernatant until the volume fraction of ethanol is 50%. The solution is then separated to obtain a second supernatant and a second precipitate. Anhydrous ethanol is continued to be added dropwise to the second supernatant until the volume fraction of ethanol is 60%. The solution is then separated to obtain a third supernatant and a third precipitate. Thus, the carboxymethyl dextran solution is divided into four molecular weight carboxymethyl dextrans corresponding to the first precipitate, second precipitate, third precipitate, and third supernatant.

3. The construction method according to claim 1, characterized in that, The cross-linking method is as follows: EDC and NHS are dissolved in 20 mM MES activation buffer, then mixed with CMG and reacted under magnetic stirring for 30 min. Then, 1.8 kPEI that has been fully dissolved is added dropwise. Tween 80 is added to the above mixed solution. The pH is adjusted to 5-6 using 1 M HCl. An appropriate amount of anhydrous ethanol is slowly added dropwise until the solution becomes turbid. The reaction is stopped after 50 min. The cross-linking reaction is terminated with 1 M TRIS-HCl at pH 8. Dialysis is performed in a dialysis bag at 8-14 K for 48 h.

4. The construction method according to claim 3, characterized in that, The volume ratio of Tween 80 to water solvent is 12:1100, and the mass ratio of EDC, NHS, CMG, and PEI is 5:5:8:

7.

5. The construction method according to claim 1, characterized in that, The specific steps for binding CMG-PEI NP to nucleic acid include: adding an aqueous solution of CMG-PEI NP to an equal mass of RNA solution, vortexing for 30 seconds, and incubating at room temperature for 30 minutes to obtain a CMG-PEI-nucleic acid complex solution, with the mass ratio of CMG-PEI NP to RNA ranging from 1:1 to 10:

1.

6. The method according to claim 5, characterized in that, The mass ratio of CMG-PEI NP to RNA is 6:1 to 7:

1.

7. A nucleic acid transport vector based on yeast β-glucan nanoparticles, characterized in that, It is prepared by the method for constructing a nucleic acid transport vector according to any one of claims 1-6.

8. The application of the nucleic acid transport vector as described in claim 7 in the transport of nucleic acids, characterized in that, The CMG-PEI NP obtained in claim 1 is compounded with RNA, and the CMG-PEI nucleic acid complex is encapsulated with mannotriose to form a nucleic acid transport vector for transporting RNA.

9. The application according to claim 8, characterized in that, The preparation of the CMG-PEI-nucleic acid gene transport vector specifically includes: mixing eGFP mRNA with CMG-PEI NP, vortexing vigorously for about 30 seconds, incubating at room temperature for about 30 minutes, adding the CMG-PEI NP and nucleic acid complex to mannotriose, with a final concentration of 4 mg / mL, mixing well, and reacting overnight for 12 hours. The mass ratio of CMG-PEI NP to eGFP mRNA is 16:1 to 48:

1.

10. The application according to claim 9, characterized in that, The mass ratio of CMG-PEI NP:eGFP mRNA is 48:1.

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